Insulin and insulin-like growth factor-1 (IGF1) are closely related polypeptide hormones/growth factors that regulate cell growth and metabolism. Insulin and IGF1 exert their biological effects on target cells by binding to distinct cell surface receptors, the insulin receptor (IR) and the IGF1 receptor (IGF1R), respectively, which are structurally related receptor tyrosine kinases. IR and IGF1R have low basal activity, which is stimulated through ligand binding to the ectodomains, resulting in autophosphorylation of specific tyrosine residues in the cytoplasmic tyrosine kinase domains. Aberrant signaling by IR and IGF1R is relevant to human disease: decreased signaling of IR plays a role in non-insulin-dependent diabetes mellitus, while increased signaling of IGF1R and IR is implicated in cancer. Despite extensive structural and biochemical studies of the separate ectodomains and cytoplasmic domains of these receptors, a comprehensive understanding of the signal transduction mechanism for IR and IGF1R is still lacking. To address these issues, we have produced highly purified preparations of full-length IR and IGF1R that are detergent-soluble, functional, and responsive to their ligands. We will use biochemical approaches and single-molecule fluorescence resonance energy transfer (FRET) to understand the relative positioning of the tyrosine kinase domains in the unliganded, basal and phosphorylated, activated states. These structure-function studies will be correlated with studies of the metabolic functions of IR and the growth-promoting effects of IGF1R in cultured cells. We will also test the hypothesis that cellular sterol and lipid composition affects transmembrane signaling by IR and IGF1R. To do this, we will take advantage of new methodology to manipulate the composition of the plasma membrane in living cells. Finally, we will characterize the interaction between IGF1R and a soluble fragment of E-cadherin (sEcad) that is shed from cancer cells. Previous work showed that sEcad activates IGF1R signaling in cultured cancer cells and in human cancer specimens, and we have shown that this is due to a direct interaction between the proteins. We will identify binding determinants on IGF1R and on sEcad that are important for this interaction. The sEcad-IGF1R interaction represents a novel target for therapeutic intervention. The work proposed in this grant application will advance our knowledge of the molecular mechanisms involved in transmembrane signaling by IR and IGF1R. This information should prove to be valuable in the design of small-molecule agonists that act intracellularly to activate IR; such agonists would have potential use as anti- diabetic therapeutics. In addition, from an understanding of the activation mechanism, it might be possible to develop IR/IGF1R inhibitors to block receptor signaling in cancer cells.